Soluble recombinant plasmodium falciparum circumsporozoite protein, use in vaccines, methods of making and uses thereof

ABSTRACT

The present invention provides novel nucleotide sequence and other constructs used for expression of novel recombinant  P. falciparum  circumsporozoite proteins in bacterial cells such as  E. coli . Processes are provided for producing a soluble recombinant  P. falciparum  CSP from  E. coli . Methods to produce a human-grade, highly immunogenic anti-malaria vaccine based on CSP are shown. The novel recombinant  P. falciparum  circumsporozoite protein by itself or in combination with other malaria antigens or adjuvants can form the basis of an effective malaria vaccine.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application from Ser. No. 13/880,227filed Apr. 18, 2013, which is a 371 national stage application from PCTApplication Serial No. PCT/US2011/056729 filed Oct. 18, 2011, entitled“Plasmodium Falciparum Circumsporozoite Vaccine Gene Optimization forSoluble Protein Expression” which claims the benefit of priority fromU.S. Provisional Patent Application Ser. No. 61/394,048 entitled“Plasmodium Falciparum Circumsporozoite Vaccine Gene Optimization forSoluble Protein Expression” filed on Oct. 18, 2010. Each application isincorporated by reference in their entireties.

RIGHTS IN THE INVENTION

The present invention was made with support from the United StatesGovernment and, specifically, the Walter Reed Army Institute ofResearch, and, accordingly, the United States government has certainrights in this invention.

TECHNICAL FIELD

The present technology is directed generally to recombinant Plasmodiumfalciparum circumsporozoite proteins, to methods for production of thoseproteins, and to vaccines including those proteins, among other aspects.

BACKGROUND

A malaria parasite infected mosquito can inject approximately 100-200Plasmodium sporozoites under the human skin during a blood meal. Thesesporozoites travel a considerable distance through several layers oftissue to reach the liver. The sporozoite journey from skin to the livercan take several minutes, during which it is exposed to the host immunesystem. Each successful sporozoite invasion can yield ˜30,000 bloodstage merozoites, each capable of invading an RBC seconds after itsrelease (Blum-Tirouvanziam, Servis et al. 1995). Hence immuneinterventions that block sporozoite invasion can be the most effectivestrategy to induce sterile immunity in humans (Blum-Tirouvanziam, Serviset al. 1995). The most abundant sporozoite surface protein of P.falciparum is the 397 amino acid long Circumsporozoite protein (CSP). Acomparison of amino acid sequences of CSP across the genus Plasmodiumrevealed a highly conserved gene structure (Doolan, Saul et al. 1992).The central region of the gene consists of a species specific repeatsequence flanked by a N-terminal region that contains a conservedstretch of a five amino acid sequence called “region I” and theC-terminal that contains a conserved cell-adhesive motif similar to onefound on thrombospondin.

The functional role of CSP in the life cycle of the parasite ismultifaceted. Genetic knockout studies with CSP show its involvement indevelopment of sporozoites from mosquito oocysts (Menard, Sultan et al.1997). CSP also binds specifically to salivary glands and is involved inthe movement of sporozoites from oocysts to the salivary glands (Wang,Fujioka et al. 2005) (Myung, Marshall et al. 2004). Genetic replacementof P. berghei CSP with the corresponding CSP from the avian malariaparasite P. gallinaceum showed a failure to invade mosquito salivaryglands and to infect mice (Tewari R, Rathore D, et al. 2005). Once onthe surface of the infective sporozoite stage, the N- and C-terminalregions of CSP have an adhesive function that along with thethrombospondin-related adhesive protein allow the sporozoite binding toheparan sulfate proteoglycans on the liver cell (Frevert 1999). CSP isalso known to shield the sporozoite as it traverses through severallayers of host tissues including professional phagocytes (Usynin, Klotzet al. 2007) and inhibits host cell protein synthesis. After invasionCSP gets exported into the hepatocyte cytoplasm and the nucleus where itcan alter the gene expression profile of the host cell to protect theparasite and promote its growth and maturation (Singh, Buscaglia et al.2007).

RTS,S is a human malaria vaccine grown in yeast cells and comprises thecentral repeats and the C-terminal cysteine rich region of Plasmodiumfalciparum CSP, fused to the S antigen of hepatitis B virus. Theexpressed protein self-assembles into a particle and is formulated withthe proprietary adjuvant system ASOX (GlaxoSmithKline, Belgium) thatcontains immune stimulants MPL and QS21 (Cohen, Nussenzweig et al.2010). Among the malaria naive individuals, RTS,S vaccination protectsapproximately 40% of vaccinees against experimental sporozoite challenge(Cohen, Nussenzweig et al. 2010). In a phase 2b trial the efficacy ofRTS,S was estimated to be ˜35% against first clinical episode and ˜49%against severe malaria during an 18-month period among 1- to 4-year-oldAfrican children (Alonso, Sacarlal et al. 2004). In another phase IItrial among 5-17 month old children, vaccine efficacy of RTS,S againstclinical episodes was found to be 53% during an average 8 month periodof observation (Bejon, Lusingu et al. 2008).

SUMMARY OF THE INVENTION

The present technology provides novel recombinant Plasmodium falciparumcircumsporozoite proteins (rCSP), along with nucleotide sequences thatexpress the recombinant P. falciparum CSP in bacterial cells, such as E.coli, as a soluble protein. The present technology also providesprocesses of expressing and purifying a soluble recombinant CSP proteinwithout denaturing or refolding the protein. The purified proteinproduced by the present technology can be greater than 95% pure (thatis, the soluble protein present in the composition is greater than 95%rCSP by weight) and contain low levels of endotoxin and low orundetectable levels of host cell proteins when analyzed by currenttechniques.

As one aspect of the present technology, novel recombinant Plasmodiumfalciparum circumsporozoite proteins are provided. The recombinant P.falciparum circumsporozoite proteins are characterized by an N-terminalregion that lacks twenty to twenty-five N-terminus amino acid residuesof native P. falciparum circumsporozoite protein; a reduced number ofNANP SEQ ID NO: 13 repeats compared to native P. falciparumcircumsporozoite protein; and at least 85% homology to SEQ ID NO: 2,alternatively at least 90% homology to SEQ ID NO: 2, alternatively atleast 95% homology to SEQ ID NO: 2. Preferably the recombinant P.falciparum circumsporozoite proteins comprise the peptide sequence ofSEQ ID NO: 2 or SEQ ID NO: 8. In some embodiments, the protein lacksMet₁ to Cys₂₅ of the N-terminal region of native P. falciparumcircumsporozoite protein. In some embodiments, the protein has 18 or 19NANP SEQ ID NO: 13 repeats, preferably 19 NANP SEQ ID NO: 13 repeats,and/or has 0 to 3 NVDP SEQ ID NO. 14 repeats, preferably 3 NVDP SEQ IDNO. 14 repeats. In some embodiments, the recombinant P. falciparum CSPhas a C-terminal region, preferably one that lacks ten to fourteenC-terminus amino acid residues of native P. falciparum circumsporozoiteprotein, more preferably, the protein ends at Ser₃₈₃.

As another aspect of the present technology, nucleotide sequences areprovided which encode a recombinant P. falciparum CSP as described inthe preceding paragraph or elsewhere in this specification. Suitablenucleotide sequences include nucleotide sequences comprising SEQ ID NO:1 or sequences that are at least 85% homologous to SEQ ID NO: 1,alternatively at least 90% homologous to SEQ ID NO: 1; alternatively atleast 95% homologous to SEQ ID NO: 1. The nucleotide sequences caninclude at least one expression tag, such as the sequence of SEQ ID NO:5.

As another aspect of the present technology, novel expression vectorsare provided for E. coli comprising a nucleotide sequence which encodesa recombinant P. falciparum CSP as described herein. The expressionvectors can be stably cloned into a bacterial cell. A suitable bacterialcell can be transformed with such an expression vector. Preferably thebacterial cell is an E. coli cell, more preferably the SHUFFLE™ strainof E. coli. (New England Biolabs, Inc., Ipswhich, Mass., described inU.S. Pat. No. 6,569,669, incorporated by reference). Surprisingly, thetransfected E. coli cell expresses a recombinant P. falciparum CSP as asoluble protein.

As yet another aspect of the present technology, anti-malaria vaccinessuitable for human administration are provided. The vaccines comprise arecombinant Plasmodium falciparum CSP as described herein, and one ormore adjuvants. Preferred embodiments of the vaccines have an endotoxinlevel less than about 5 endotoxin units per microgram of protein, and/orless than about 1 ng/ml of bacterial host proteins. In some embodiments,the vaccines have a soluble protein content, and the soluble proteincontent is greater than 95%, alternatively greater than 99%, purerecombinant P. falciparum CSP as measured by gel densitometry.

As another aspect of the present technology, methods of eliciting animmune response against malaria in an animal or human compriseadministering a vaccine or rCSP as described herein. Methods ofimmunizing an animal or human against malaria or a pathogen that causesmalaria are also provided. The methods comprise administering to theanimal or human a vaccine or rCSP as described herein. In these methods,the vaccine can be administered intramuscularly or by another route.

As still another aspect of the present technology, processes ofproducing recombinant P. falciparum CSP are provided, includingprocesses of producing rCSP in soluble form from E. coli. The processescomprise the steps of providing cells, preferably bacterial cells suchas E. coli, containing a nucleotide sequence that expresses one of therecombinant P. falciparum CSPs described herein (such as a transfectedE. coli that expresses the peptide sequence of SEQ ID NO: 2). The cellsmay be provided in a cell culture. The processes also comprise inducingexpression of the recombinant CSP in the cells, and collecting the cellsafter a period of expression, such as by centrifuging to obtain a pelletcontaining the cells. The processes also comprise lysing the cells toobtain a cell lysate, collecting supernatant from the cell lysate, andpurifying the recombinant P. falciparum CSP from the supernatant of thecell lysate without denaturing and refolding the recombinant P.falciparum CSP. Preferably, the bacterial cell is cultured in that ismedia free or substantially free of animal-derived components, such asmedia containing one or more or all of Phytone, yeast extract, ammoniumsulfate, potassium phosphate monobasic, sodium phosphate dibasic, MgSO₄,glycerol, dextrose or kanamycin. In some embodiments, the recombinant P.falciparum CSP includes one or more expression tags at one or both endsto facilitate the recovery or purification of the protein. The processescan include one or more purification steps, such as purifying thesoluble protein over an affinity column and purifying the solubleprotein over an anion exchange column, for example, a nickel affinitycolumn and a Q-sepharose anion exchange column. The present technologyalso includes purified protein made by the processes described herein.

In preferred embodiments of the production processes, purified proteinis recovered from a two-step purification procedure over the affinitycolumn and the anion exchange column, and the purification proceduredoes not include any other chromatographic separation or consistsessentially of an affinity column separation on an anion exchange columnseparation. Alternatively, the production process has no more than twopurification steps. The purified protein of the present technology cancontain at least about 90% recombinant P. falciparum CSP, alternativelyat least about 95% or at least about 99%, as measured by geldensitometry. The processes can also include the step of filtering thepurified protein. The protein can contain less than 1 ng/ml of E. colihost proteins and/or less than about 5 endotoxin units per microgramprotein. Preferably the production process meets or exceeds current goodmanufacturing practices (for example, as described in the US Code ofFederal Regulations, Title 21) for vaccine products. The presenttechnology also includes a vaccine comprising a purified proteinproduced by the foregoing process.

As another aspect, the present technology provides novel CS geneconstructs that encode an amino acid sequence having a start site atTyr₂₆, 19 copies of the NANP SEQ ID NO: 13 amino acid repeat, and 3copies of the NVDP SEQ ID NO. 14 repeat. On the C-terminal region, theglycosylphosphatidylinositol (GPI) anchor sequence can be excluded fromthe CS gene construct and excluded from the rCSP; in other words the CSgene construct does not include a nucleotide sequence that encodes theGPI anchor sequence, and the rCSP does not include the GPI anchorsequence. The novel soluble recombinant proteins of the presenttechnology can comprise a protein sequence with at least 85% homology toSEQ ID NO: 2, preferably 90% homology to SEQ ID NO: 2, preferably atleast 95% homology to SEQ ID NO: 2, more preferably about 99% homologyto SEQ ID NO: 2, most preferably includes SEQ ID NO: 2 and in someembodiments include at least one expression tag, preferably at least twoexpression tags. In some aspects, at least one expression tag is ahistidine tag, for example, a 6×HIS tag, preferably two 6×HIS tags, suchas in SEQ ID NO: 8. Novel nucleotide sequences are provided that encodethe soluble CSPs of the present technology, which can be expressed in E.coli and have at least 85% homology, preferably at least 90% homology,preferably at least 95% homology, more preferably at least 99% homologyto SEQ ID NO: 1. In some aspects, the nucleotide sequence encoding theCSP of the present technology comprises nucleotide sequences for atleast one expression tag, preferably at least two expression tags (forexample, expression tags SEQ ID NO: 3 and SEQ ID NO: 4), and includesSEQ ID NO: 5.

In another aspect, the present technology provides vaccines comprisingthe soluble rCSP disclosed herein and at least one adjuvant. Thevaccines can be used to vaccinate a subject (such as a human or animal)and elicit an immune response. In some aspects, the vaccine produceshigh titer antibodies in the subject.

In some aspects, the novel rCSP induces high titer antibodies whenformulated with at least one adjuvant, preferably Montanide ISA 720adjuvant (Seppic Inc, France). In further aspects, vaccination with thesoluble protein of the present technology and at least one adjuvantconfers partial or full protection in a vaccinated subject against amalaria challenge, and in some aspects provides sterile protectionagainst malaria challenge.

In another aspect, anti-rCSP antibodies produced after immunization withthe soluble protein of the present technology can also recognize nativeCSP on sporozoites. The present technology includes those anti-rCSPantibodies.

In some aspects, the final recombinant CSP protein of the presenttechnology is of high purity and suitable for human vaccination againstmalaria. Further, the protein of the present technology can be producedunder current good manufacturing practices to produce a vaccine gradeprotein composition made in animal-free media, a media free ofanimal-derived components. A human-grade vaccine suitable foradministration to human subjects can be produced.

The purified rCSP product of the present technology meets the puritycriteria for an injectable for human administration (>95% purity by geldensitometry, low or undetectable levels of host cell proteins, asdetected by western blot, and less than 5 endotoxin units/microgramtotal protein. Further, the rCSP protein product is structurallyhomogeneous as observed on reduced and non-reduced SDS-page and isstable at 4° C. for at least a week.

In some aspects, the novel rCSP of the present technology is stronglyimmunogenic and provides protection against challenge of sporozoites.

In some aspects, the present technology provides a method of producing anear full-length soluble CSP (such as the peptide sequence of SEQ ID NO:2) that is stable at high concentrations in aqueous buffer and suitablenucleotide sequences for producing the soluble protein in E. coli.

In a further aspect, the present technology provides a PfCSP genenucleotide sequence that in combination with an E. coli host strainproduce correctly folded and soluble CSP without requiring a denaturingand refolding step in the production process of the proteins.

In yet a further aspect, the present technology provides a fermentationprocess that promotes the growth of a CSP-producing bacterial clone innon-animal derived media.

In still another aspect, the present technology provides a two-steppurification process which results in a greater than 95% pure product ofsoluble recombinant CSP with low endotoxin levels (for example, lessthan 5 endotoxin units per microgram) and low or undetectable levels ofhost proteins.

In yet another aspect, the present technology provides an E. coli hostcell that produces a soluble recombinant CSP that can be used forvaccination against malaria. The vaccine containing the CSP can offerimproved immunogenicity over yeast derived CSP vaccines which may bepost-translationally modified, truncated or glycosylated. Thus, in someembodiments, the present vaccines comprise rCSP that is not glycosylatedor post-translationally modified or truncated.

In another aspect, the present technology provides a vaccine that can beeconomically produced by expression in E. coli and a two-steppurification system that results in a substantially pure solubleprotein.

Other aspects and advantages of the present technology are set forth inpart in the description, as follows, and in part, may be obvious fromthe description, or may be learned from practice of the presenttechnology.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a cartoon representation of the protein expression constructsCS/A, CS/B, CS/C, CS/D, and CS/E shown in relation to native P.falciparum CSP gene 3D7 strain (top). The number of NANP SEQ ID NO: 13and NVDP SEQ ID NO. 14 repeats and relative position of the cysteineresidues (C) are also shown (figure not drawn to scale).

FIGS. 2A, 2B and 2C depict the thermal stability of CS/A and CS/Bsoluble protein. FIG. 2A shows purified CS/A (lane 1) and CS/B (lane 2)protein as analyzed by SDS-PAGE via coomassie blue staining and westernblot analysis using an anti-6×HIS monoclonal antibody after onefreeze-thaw cycle. FIG. 2B shows an image of precipitated CS/A proteinhaving a fibrillar structure upon negative staining and electronmicroscopy imaging. FIG. 2C demonstrates thermal stability of CS/Bprotein following storage for one week at 37° C. (lane 1), 4° C. (lane2) and −80° C. (lane 3) as demonstrated by SDS-PAGE and coomassie bluestaining.

FIG. 3 illustrates the comparative expression levels of CS/C, CS/D, andCS/E in E. coli. Uninduced (U) and IPTG induced (In) whole E. coli cellpellets were analyzed by SDS-PAGE and stained by coomassie blue. CS/C (5NANP SEQ ID NO: 13 repeat containing), CS/D (19 NANP SEQ ID NO: 13repeat containing) and CS/E (38 NANP SEQ ID NO: 13 repeat containing)proteins are shown by the arrows.

FIGS. 4A, 4B and 4C illustrate the immunogenicity of rCSP proteins basedon the number of NANP SEQ ID NO: 13 repeats. Groups of mice (9 pergroup) were immunized with either CS/C (a 5 NANP SEQ ID NO: 13 repeatrCS protein) or CS/D (a 19 NANP SEQ ID NO: 13 repeat CS protein).End-point ELISA titers were determined for the mice two weeks afterimmunization with the second dose of either the CS/C or CS/D protein.Antibody titers were measured using ELISA plates coated with CS/C (FIG.4A), CS/D (FIG. 4B), or a 6×NANP SEQ ID NO: 13 repeat peptide (FIG. 4C).

FIG. 5 depicts the nucleotide sequence of the recombinant CSP gene ofthe present technology with and without two expression 6×HIS tags.

FIG. 6 depicts the translated protein sequence of the soluble CSP andthe N and C-terminal tags.

FIGS. 7A, 7B, 7C, and 7D depict the results of purity and identitystudies on a laboratory grade CS/D protein. FIG. 7A is a SDS-PAGEanalysis of the Ni-NTA Ni affinity elution and Q-sepharose elutionstained by coomassie blue. FIG. 7B is the SDS-PAGE analysis of theelution from a Superdex-75 gel filtration column stained with coomassieblue. FIG. 7C is SDS-PAGE analysis of the CS/D purified protein with 2μg, 1 μg, 0.5 μg, 0.25 μg and 0.1 μg protein loaded per lane (lanes 1-5respectively) analyzed under non-reducing and reducing conditions andvisualized by silver staining (Snap Silver Kit™, Pierce). FIG. 7D is ananti-CSP mAb Western Blot showing 1 μg CS/D protein positively reactingwith the anti-P. falciparum CSP specific mAb 49-1B2 (left panel, lane1). Western blot was also performed to determine purity of the proteinusing an anti-E. coli polyclonal antibody (Dako Corp). Lane 1 and 2contain 1 μg and 2 μg purified CS/D protein respectively and lane 3contains the lysate of bacteria (right panel).

FIGS. 8A and 8B depict the immunogenicity and protection efficacy oflab-grade CS/D shown in FIG. 7. FIG. 8A shows specific end-point ELISAtiters against CS/D protein coated plates. Titers of individual CSPvaccinated mice were plotted against time (end point defined as serumdilution that results in an OD=0.5). Days of vaccination are shown byarrows. FIG. 8B shows the survival curve of the CSP vaccinated andcontrol group of mice following challenge with 5,000 transgenicsporozoites (Tewari, Spaccapelo et al., 2002). FIG. 8C demonstrates theantibody reactivity of CSP vaccinated mouse sera with methanol fixedsporozoites using an immuno-fluorescence assay, showing positivereactivity of anti-CS antibodies in the fluorescence field (right).

FIG. 9 depicts the growth curve of bacteria containing a CSP constructin a 300 L fermentor produced under cGMP environment at theBioproduction facility at the Walter Reed Army Institute of Research,Silver Spring, Md.

FIG. 10 depicts SDS-PAGE analysis using coomassie blue staining ofsamples collected during the cGMP compliant purification process of thepresent technology, including protein that was loaded on the Ni column(lane 1), wash samples with buffer A (lane 2), wash samples with bufferB (lane 3), wash samples with buffer C (lane 4), wash samples withbuffer D (lane 5), elution sample from Ni column (lane 6), sample thatflowed through Q column (lane 7), buffer wash F (lane 8), buffer wash G(lane 9), buffer wash F (lane 10), buffer wash H (lane 11), sampleeluted from Q column (lane 12) and post filtration CS/D bulk (lane 13).

FIG. 11 depicts the purity of a sample of purified soluble protein rununder non-reduced and reduced conditions on SDS-PAGE by Coomassie bluestaining.

FIG. 12 depicts the purity of the cGMP compliant purified protein usingsilver staining under reducing conditions.

FIGS. 13A and 13B depict the stability of the cGMP grade soluble proteinof the present technology during a freeze-thaw cycle. Samples wereanalyzed after thawing (labeled as ‘thawed’) and after a spin at 10K for10 min (labeled as ‘thawed and spun’) on SDS-PAGE (A) and western blot(B) using anti-CSP antibodies under reduced (Red) or non-reduced (NR)conditions. The anti-CSP antibodies specifically bind to CSP.

FIG. 14 depicts the thermal stability of cGMP grade CS/D purifiedsoluble protein analyzed by non-reduced coomassie stained SDS-PAGE atfour different temperatures and three time points.

FIG. 15 depicts the reversed-phase HPLC profile of CS/D soluble purifiedprotein.

FIG. 16 depicts the antibody titers of individual mice between thesecond and third boosting doses of the cGMP grade soluble protein of thepresent technology.

FIGS. 17A and 17B depict the survival curves of mice in the cGMP rCSPCS/D vaccine (A) and adjuvant control (B) groups. Mice that did notbecome infected by blood stage parasites were considered as protected.

FIG. 18 depicts the correlation between NANP SEQ ID NO: 13 repeatspecific ELISA titer (x axis) and full-length protein ELISA titer (yaxis).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following detailed description, for purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various embodiments of the present invention. Itwill be apparent, however, that the various embodiments of the presentinvention may be practiced without these specific details.

The present technology provides an E. coli-produced soluble recombinantCSP and a process to manufacture this protein in a cGMP (current GoodManufacturing Practice, in compliance with CFR Title 21) compliantenvironment for use as a component of an anti-malarial vaccine. CurrentGMP for use of products as human injectables is outlined by the Food andDrug Administration in CFR Title 21. For a protein to be used as a humaninjectable, it has to meet requirements of purity and be grown inconditions that provide a product substantially free of othercontaminants.

Expression of the P. falciparum CSP gene in E. coli has remained a majorchallenge. The native gene has an AT content of ˜65% and it containsseveral codons for which the cognate tRNA's in E. coli are in lowabundance (including 6×GGA, 5×AUA, 1×CUA, 3×AGA and 2×AGG codons). Thehigh AT content of PfCSP and the presence of rare codons has made itextremely difficult to express PfCSP in E. coli. The complex primary andtertiary structure of CSP pose further problems with prokaryoticexpression. This includes two disulphide bonds in the C-terminal regionand a highly repetitive NANP SEQ ID NO: 13 sequence in the middle.Proteins expressed in conventional E. coli strains do not form correctdisulphide bonds due to the reducing cytosolic environment. The 38 NANPSEQ ID NO: 13 repeats of PfCSP cause extensive genetic rearrangementduring the gene cloning and plasmid maintenance. It is not surprisingtherefore that expression of a soluble PfCSP in E. coli has apparentlynot been accomplished previously and thus only insoluble proteinexpression of P. falciparum CSP has been reported, that requiresextensive in vitro refolding (Kolodny, Kitov et al. 2001, Plassmeyer,Reiter et al. 2009). Another problem with expressing a near full-lengthCSP is that the N-terminal processing site of native PfCSP is not known,which makes it difficult to determine a suitable start site for thefull-length PfCSP based construct for protein expression.

In the literature there are 3 reports of attempted E. coli expression offull-length Plasmodium falciparum circumsporozoite protein (CSP). Thefirst report showed that full-length CSP gene cannot be expressed in E.coli (Young, Hockmeyer et al. 1985). Two subsequent reports showedexpression of an insoluble protein product that required extensiverefolding to gain solubility (Kolodny, Kitov et al. 2001; Plassmeyer,Reiter et al. 2009). The present technology provides a novel PfCSP genesequence that yields a highly immunogenic and near full-length solublerecombinant P. falciparum circumsporozoite protein (rPfCSP) expressed inE. coli. The final rCSP protein is of high purity and suitable for humanvaccination against malaria.

In some embodiments, the present technology provides an expressionconstruct for the production of soluble recombinant P. falciparumcircumsporozoite protein which comprises the peptide sequence SEQ ID NO:2, or a peptide sequence with at least 90% homology to SEQ ID NO: 2,more preferably at least 95% homology, more preferably at least 99%homology.

Purification of the expressed protein can be achieved by any suitablemeans, such as by affinity chromatography using expression tags on therecombinant P. falciparum CSP or using antibodies that recognize theappropriate regions of the rCSP. For example, purification by affinitychromatography can be facilitated by including at least one expressiontag, preferably at least one polyhistidine tag, e.g. 6×HIS tag, in therecombinant P. falciparum CSP. In some preferred embodiments, therecombinant P. falciparum CSP includes two expression tags, for example,two polyhistidine tags on one or both of the C-terminus and/orN-terminus of SEQ ID. NO:2. Preferred embodiments of the presenttechnology include the peptide sequence of SEQ ID NO: 8, or a peptidesequence that includes the polyhistidine tags and is at least 85%similar to SEQ ID NO: 8, preferably at least about 90% similar to SEQ IDNO: 8, more preferably about 95% or more similar to SEQ ID NO: 8, whereSEQ ID NO: 8 is peptide sequence of SEQ ID NO: 2 further containing two6×HIS tag amino acid sequences, one tag on each of the N and C terminus(SEQ ID NO: 6 and 7, respectively). The present technology envisionsthat other expression tags or HIS tag sequences may be used inaccordance with the present technology in combination with peptidesequence of SEQ ID NO: 2 to produce similar or equivalent results asdemonstrated with the soluble protein described herein. The expressiontags may be added either at the N-terminus or C-terminus or both.Suitable expression tags include, but are not limited to, myc tag, flagtag, and the like. The total number of histidine residues may vary inthe tag. The tag may also be preceded or followed by a suitable aminoacid sequence that facilitates a removal of the polyhistidine-tag usingendopeptidases. Suitable peptide sequences for N terminal 6×HIS taginclude, but are not limited to, SEQ ID NO: 6 and suitable peptidesequences for a C-terminal 6×HIS tag include, but are not limited to,SEQ ID NO: 7.

In some embodiments, the present technology provides novel nucleotidesequence that encodes the soluble CSP of the present technology whichcan be expressed in E. coli and has at least 85% homology, alternativelyat least 90% homology, preferably 95% homology, more preferably 99%homology to SEQ ID NO: 1.

In some embodiments, the nucleotide sequence encoding the CSP of thepresent technology further includes at least one nucleotide sequence,preferably two nucleotide sequences encoding at least one expression tagsequence, preferably at least two expression tag sequences, suitably oneor more polyhistidine tag sequences. Suitable polyhistidine sequencesinclude, but are not limited to, 6×HIS, including SEQ ID NO: 3 and SEQID NO: 4. Other suitable expression tag or 6×HIS tag sequences known inthe art may be used and are contemplated to be used to express andpurify a protein with similar or equivalent characteristics as describedin the present technology. The expression tag sequence may be added tothe N terminus, C terminus or both of the rCSP gene sequence. Apreferred nucleotide sequence of the present technology includes asequence with at least about 85% homology to SEQ ID NO: 5, preferably atleast about 90% homology, more preferably at least about 95% homology toSEQ ID NO: 5 and includes SEQ ID NO: 5, which is the combination of SEQID NO: 1 with a N-terminal HIS tag (SEQ ID NO: 3) and C-terminal HIS tag(SEQ ID NO: 4). A suitable nucleotide sequence of the present technologyincludes SEQ ID NO: 1 or a nucleotide sequence that is at least 85%similar to SEQ ID NO: 1, preferably about 90% similar to SEQ ID NO: 1,more preferably at least about 95% similar to SEQ ID NO: 1 and includesat least one expression tag sequence, preferably at least two expressiontag sequences, most preferably HIS tag sequences.

In some embodiments, the nucleotide sequence of the present technologymay be cloned into a suitable expression vector for expression in E.coli. Preferably, a nucleotide sequence of the present technology, orfor use in the present technology in a vector, is operably linked to acontrol sequence which is capable of providing for the expression of thecoding sequence by the host cell, in other words, the vector is anexpression vector. The term “operably linked” refers to a juxtapositionwherein the components described are in a relationship permitting themto function in their intended manner. A regulatory sequence, such as apromoter, “operably linked” to a coding sequence is positioned in such away that expression of the coding sequence is achieved under conditionscompatible with the regulatory sequence.

Suitable expression vectors are known in the art and include, but arenot limited to, plasmids, for example, pET plasmid (Novagen, Merck,Whitehouse Station, Mass.) or the pQE plasmids (Qiagen, Valencia,Calif.). The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene or kanamycin resistance genein the case of a bacterial plasmid. Promoters and other expressionregulation signals may be selected to be compatible with the host E.coli cell for which expression is designed to be used. All thesepromoters are well described and readily available in the art.

In further embodiments, the present technology provides a bacterialcell, such as an E. coli cell, transformed with one of nucleotidesequences described above, preferably a nucleotide sequence comprisingSEQ ID NO: 1, more preferably comprising SEQ ID NO: 5. Preferably thebacteria is E. coli, and in the preferred embodiments, the E. colistrain is the SHUFFLE™ strain.

In another embodiment of the present technology provides a human-gradeanti-malaria vaccine. The anti-malaria vaccine comprises a solublerecombinant P. falciparum circumsporozoite protein of the presenttechnology. Preferably the vaccine comprises at least one adjuvant andthe soluble recombinant protein contains SEQ ID NO: 2 and at least onetag sequence, more preferably two expression tags, for example two 6×HIStags (e.g. SEQ ID NO: 8). A human dose of the soluble rCSP can bebetween about 1 to about 100 micrograms, and the concentration ofadjuvant can be determined by one skilled in the art. The rCSP is“soluble” in that it is expressed from a cell, preferably a bacterialcell, without the need for denaturing and refolding. It may or may notbe soluble in the vaccine; that is, the vaccine may be a solution, aparticle, or a suspension of the rCSP.

The vaccine or purified protein of the present technology comprise lowlevels of endotoxin, preferably less than about 5 endotoxin units (EU)per microgram protein as measured by chromogenic Limulus amebocytelysate (LAL) endpoint assay (Associates of Cape Cod, Falmouth, Mass.).See e.g., Bacterial Endotoxins Test, United States Pharmacopeia (currentrevision), United States Pharmacopeial Convention, Rockville, Md. Insome embodiments, the vaccine or protein composition of the presenttechnology comprises less than about 2 EU/μg protein, alternatively lessthan about 1 EU/μg protein, alternatively less than about 0.5 EU/μgprotein. In some embodiments, undetectable levels of endotoxin are foundin the vaccine or purified protein, for example, less than about 0.1EU/μg protein by LAL assay. Other suitable assays are known in the artand include, for example, the Gel-clot assay, described in the examplesbelow.

Vaccines of the present technology typically include at least oneadjuvant. Suitable adjuvants include, but are not limited to, aluminumsalts such as aluminum hydroxide or aluminum phosphate, salts ofcalcium, iron or zinc, insoluble suspensions of acylated tyrosine, oracylated sugars. Other suitable adjuvants cationically or anionicallyderivatized saccharides, polyphosphazenes, biodegradable microspheres,nanoparticles, liposome based formulations, monophosphoryl lipid A(MPL), lipid A derivatives (for example, of reduced toxicity),3-O-deacylated MPL, quil A, Saponin, QS21, Freund's Incomplete Adjuvant(Difco Laboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck andCompany, Inc., Rahway, N.J.), emulsion or a water-in-oil emulsion, ASO(Smith-Kline Beecham, Philadelphia, Pa.), AS)1 (GlaxoSmithKline), CpGoligonucleotides, bioadhesives and mucoadhesives, polyoxyethylene etherformulations, polyoxyethylene ester formulations, muramyl peptides orimidazoquinolone compounds (e.g. imiquamod and its homologues), orMontanide ISA 720. Human immunomodulators suitable for use as adjuvantsin the invention include cytokines such as interleukins (e.g. IL-1,IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc), macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), granulocyte and macrophagecolony stimulating factor (GM-CSF). The adjuvant may be provided in theform of microparticles or liposomes containing one or more of theadjuvants disclosed herein or other adjuvants, either inside theparticle or on the surface. Alternatively, some adjuvants can beprovided in the form of an oil and water emulsion, such as anoil-in-water emulsion or a water-in-oil emulsion. In a preferredembodiment, the adjuvant of the present technology is Montanide ISA 720.In some embodiments, the adjuvant can be selected to induce a specifictype of immune response, such as a B-cell response or a T-cell response.In one embodiment of the present technology, the vaccine induces animmune response. Suitable adjuvants which promote an appropriate immuneresponse include, but are not limited to, derivatives of lipid A(preferably of reduced toxicity), Monophosphoryl lipid A (MPL) or aderivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A(3D-MPL), and a combination of monophosphoryl lipid A, optionally3-de-O-acylated monophosphoryl lipid A together with an aluminum salt.

In some embodiments, the vaccines of the present technology, in additionto the rCS protein and at least one adjuvant, comprise one or morepharmaceutically acceptable carriers or excipients. Excipients includeany component that does not itself induce the production of antibodiesand is not harmful to the subject receiving the composition. Suitableexcipients are typically large, slowly metabolized macromolecules suchas proteins, saccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, sucrose, trehalose,lactose and lipid aggregates (such as oil droplets or liposomes).Suitable pharmaceutical carriers are well known to those of ordinaryskill in the art, including, but not limited to, diluents, such aswater, saline, glycerol, and others. Suitably, sterile pyrogen-free,phosphate buffered physiologic saline is a pharmaceutical carrier.Additionally, additives, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present. Vaccines of thepresent technology are formulated into suitable dosage for the subjectto which it is to be administered. The dosage administered may vary withthe condition, sex, weight and age of the individual; the route ofadministration; and the adjuvant used. The vaccine may be used in dosageforms such as suspensions or liquid solutions. The vaccine may beformulated with an pharmaceutically acceptable carrier as describedabove. Suitable dosages include, but are not limited to, about 1 toabout 100 micrograms, alternatively about 5 to about 50 micrograms, of arecombinant CSP described herein.

The present technology provides a method for raising an immune responsein a subject, comprising the step of administering an effective amountof a vaccine of the present technology. The vaccines can be administeredprophylactically (i.e. to prevent infection) or to provide protectiveand preferably involves induction of antibodies and/or T cell immunityagainst CSP. The method may raise a primary immune response, a secondaryimmune response, a booster response or a combination of immuneresponses.

Subjects may receive one or several booster (subsequent) immunizationsadequately spaced. Dosing treatment can be a single dose schedule or amultiple dose schedule. Multiple doses may be used in a primaryimmunization schedule and/or in a booster immunization schedule.Suitable timing between the administration of priming doses (e.g.between 4-16 weeks) and between the administration of priming andboosting doses can be determined.

Vaccines of the present technology may be in an aqueous form, forexample, but not limited to, solutions, particles or suspensions. Thevaccine can be an oil and water emulsion, such as an oil-in-wateremulsion or a water-in-oil emulsion. Liquid formulations allow thecompositions to be prepackaged and administered direct from theirpackaged form without the need for reconstitution. Compositions may bepresented in vials, or they may be presented in ready filled syringes. Asyringe can include a single dose of the composition, whereas a vial mayinclude a single dose or multiple doses (e.g., 2, 3, 4, 5, 10, or moredoses). Preferably, the dose is for human administration, suitably foran adult, adolescent, toddler, infant or less than one year old humanand may be administered by injection.

The vaccines or compositions of the present technology can beadministered by a variety of routes, including, but not limited to,orally, parenterally, subcutaneously, mucosally, intravenously orintramuscularly.

In some embodiments, the vaccine of the present technology comprisesrecombinant CSP protein and has low or undetectable levels of host cellproteins, preferably less than about 1 ng/ml of host cell proteins. Insome embodiments, the amount of host cell proteins would be anundetectable amount, as measured by SDS-PAGE or host cell protein (HSP)ELISA or western blots using antibodies against host cell proteins.

In some embodiments, the soluble protein of the present technology isgreater than 95% pure recombinant P. falciparum CSP as measured by geldensitometry, preferably at least about 98% pure, more preferably atleast about 99% pure recombinant P. falciparum CSP as measured by geldensitometry and as evidenced by a single peak by reversed-phase HPLC.

In some embodiments, a combination vaccine comprising the recombinant P.falciparum CSP of the present technology and at least one adjuvant iscombined with at least one additional malaria antigen. Other suitablemalaria antigens are known, including without limitation, for example,blood stage antigens, liver stage antigens, sexual stage antigens,antigens expressed on RBC surface, sporozoite stage antigens andsecreted malaria antigens including MSP1, MSP2, MSP3, AMA1, LSA1, Pfsantigens, RON2, and others.

The present technology also provides methods of eliciting an immuneresponse against malaria or malaria antigens in a subject, preferably ananimal. The method includes administering to the subject a vaccinecomprising the rCSP of the present technology. An immune responseincludes, but is not limited to, antibody production, killer T-cellresponses, and helper T-cell (T_(H)) responses.

In other embodiments, the present technology provides a method ofimmunizing an animal or human against malaria by administering to theanimal the vaccine of the present technology. In some embodiments,immunizing the animal against malaria provides partial or fullprotective immunity against malaria parasite infection. Preferably thesubject is an animal, preferably a primate, more preferably a human.

In other embodiments, the present technology provides a method ofproducing a recombinant protein of P. falciparum circumsporozoite of thepresent technology in E. coli. The recombinant CSP is found producedintracellularly within the E. and can be purified from supernatant oflysed E. coli cells expressing the construct. The method includesculturing E. coli containing the nucleotide construct comprising thenucleotide sequence of the present technology in non-animal media,pelleting the E. coli cells from the E. coli culture (bycentrifugation), lysing the E. coli cells, collecting the supernatantfrom the E. coli lysate, and purifying the soluble protein from thesupernatant of the E. coli lysate without denaturing and refolding theprotein.

In some embodiments, the E. coli culture is grown using a fermentationprocess. The E. coli culture can be induced to express the protein usingIPTG, preferably at about 0.1 to about 5 mM.

In some embodiments, the purified protein or the vaccine has a host DNAcontent that is less than or equal to 2 pg per 20 micrograms of purifiedprotein. In some embodiments, the purified protein or the vaccine has aSarcosyl content below 0.0001%. In some embodiments, the purifiedprotein or the vaccine has a nickel content that is less than or equalto 0.75 micrograms per gram of purified protein or vaccine. In someembodiments, the purified protein or the vaccine has an imidazolecontent less than 50 nanomoles/mL. In some embodiments, the purifiedprotein or the vaccine has an IPTG content less than or equal to 0.05micrograms/mL.

In some embodiments, the soluble protein is purified by a two-stepprocess including affinity column and anion exchange column. The solubleprotein is purified by flowing the soluble protein over an affinitycolumn, for example a Ni-affinity column and then eluting the boundprotein from the column and then flowing the first elutant of solubleprotein over a Q-sepharose anion exchange column. Suitable affinitycolumns include, but not limited to, Ni-NTA affinity columns, cationexchange, anion exchange, tag affinity columns, and gel-filtrationcolumns. Suitable buffers for washing and elution over the affinity andsepharose columns are known to one skilled in the art, and include thebuffers as described in the examples below. The protein eluted after thetwo step purification is at least 95% pure, more preferably at leastabout 98% pure, more preferably greater than about 98% pure asdetermined by gel densitometry. The eluted purified protein also hasless than 5 EU/ml of endotoxin, preferably less than about 1 EU/ml asmeasured by the LAL assay and less than 1 ng/ml of host cell proteins asdetected by HCP (host cell protein) ELISA.

In some embodiments, the purification includes an additional step of gelfiltration. Suitable methods of gel filtration, include, but are notlimited to, Superdex™-75 column.

Purified protein may be filtered to provide a sterile product, forexample filtered through a cellulose or PVDF based filter, for example,an about 0.2 to about 0.45 micron cellulose filter, preferably a 0.22micron cellulose filter.

The purified protein of the present technology is stable in aqueoussolutions and can be stored for at least a week at 4° C. The purifiedprotein can also be concentrated to at least 0.5 mg/ml, alternativelyabout between about 1 to about 2 mg/ml.

Example 1

In this Example, the development and expression of a soluble recombinantPfCSP is described. A suitable start site for the recombinant PfCSP wasdetermined as follows. The P. falciparum CSP gene contains a signalsequence, followed by the N-terminal region, a region containing 38 NANPSEQ ID NO: 13 repeats and 4 NVDP SEQ ID NO. 14 repeats, a C-terminalcysteine-rich region and a glycophosphotidyl inositol (GPI) anchorsequence. On sporozoites, the native CSP is proteolytically processedwithin the N-terminal region, however the exact N-terminal processingsite is unknown. Not to be bound by any theory, but antibodies to a rCSPthat includes the processing site may help block the processing stepthat may play a role in the invasion step required from entry andpropagation. Hence, it was hypothesized that a portion of the N terminalregion of CSP should be retained in the PfCSP construct of the presenttechnology.

In the absence of the processing site data, two different constructs(namely, CS/A, see SEQ ID NO: 9, FIG. 1 and CS/B, see SEQ ID NO: 10,FIG. 1) were tested for expression in E. coli. P. falciparum 3D7 strainCSP gene (accession number XP001351122) was used as a template for thesesequences. It encodes native PfCSP. The CS/A construct initiated at CSPspecific residue Gln₂₁ contains 18 of the NANP SEQ ID NO: 13 repeats andends at amino acid Leu₃₈₇ (lacking the C-terminal GPI anchor sequence).CS/B only differed from CS/A at the starting residue which was Tyr₂₆.CS/A and CS/B were both cloned into pET plasmid, expressed in bacteria,and purified on a Nickel affinity column Both constructs included Histags on the N-terminus. The purified proteins were tested for stabilityby freezing and thawing the protein. After a single freeze-thaw cyclethe CS/A protein, which included the residue Cys₂₅ of native CSP, formeda high molecular weight aggregate that was visualized as a smear on theanti-6His mAb western blot (FIG. 2A, lane 1). CS/A protein solution alsoshowed a visible precipitate that had a fibrillar structure whenobserved under an electron microscope (FIG. 2B). A majority of the CS/Bexisted as a monomer after the freeze-thaw (FIG. 2A, lane 2) and onlyshowed the discrete high molecular weight bands corresponding to dimer,trimer and multimers. CS/B was further analyzed for thermal stabilityover 7 days at 37° C., 4° C. and −80° C. Samples were analyzed using SDSPage and coomassie blue staining. CS/B was found to be stable insolution at 4° C. for at least a week (FIG. 2C, lane 2), while breakdownwas observed at 37° C. (FIG. 2C, lane 1). The CS/A protein could only beconcentrated to 0.5 mg/ml using ultra-filtration, while the CS/B proteincould be concentrated to >1 mg/ml without precipitation. Thus residueCys₂₅ is preferably excluded from the recombinant PfCSP and Tyr₂₆ ispreferred as the starting amino acid residue of the recombinant PfCSP.

Example 2

The number of NANP repeats in the recombinant PfCSP was determined asfollows. The native P. falciparum CSP contains 38 NANP SEQ ID NO: 13 and4 NVDP SEQ ID NO. 14 repeats. The presence of a large number ofrepeating amino acid units makes it difficult to express CSP inheterologous expression systems. A comparison of the effect of NANP SEQID NO: 13 repeat length on the immunogenicity and expression levels ofCSP in E. coli was performed. Two different CSP constructs (namely,CS/C, see SEQ ID NO: 11, FIG. 1 and CS/D, see SEQ ID NO: 8, FIG. 1) weretransformed and expressed in E. coli. Construct CS/C and CS/D hadidentical N-termini starting at amino acid Tyr₂₆ and ending at Ser₃₈₃,the only difference between the two constructs was the number ofrepeats. CS/C contained 5 NANP SEQ ID NO: 13 and 2 NVDP SEQ ID NO. 14repeats, while CS/D contained 19 NANP SEQ ID NO: 13 and 3 NVDP SEQ IDNO. 14 repeats. The proteins were expressed in small scale cultures andthe level of expression was compared by running the whole cell lysate ofun-induced and IPTG induced bacterial pellets on an SDS PAGE. Therecombinant protein were visualized using coomassie blue staining. (FIG.3). The expression level of the CS/C was found to be the higher thanCS/D (FIG. 3) by densitometric analysis with background proteinintensity used to normalize the protein load.

The CS/D and CS/C proteins were purified by two steps of columnchromatography, and the purified proteins were then used to vaccinate agroup of mice using Montanide ISA 720 adjuvant. Two separate dosescontaining 2.5 micrograms of purified CS/C or CS/D were administered,intra-peritoneally into two groups of 9 mice, 2 weeks apart. Two weeksafter the second dose, the sera in mice were collected and were testedfor anti-CSP antibodies. Both groups of vaccinated mice (the CS/C groupand CS/D group) were found to have similarly high titers of anti-CSPantibodies when tested against whole proteins coated on the plate (FIGS.4A and 4B). However, the CS/D group titers were significantly higherthan the CS/C titers when tested against a repeat peptide ‘(NANP)₆’ SEQID NO: 13 coated on plates (p=0.0002, FIG. 4C), indicating thatincreasing the number of repeats present in the vaccine proteinincreases repeat-specific antibody production. Thus, the proteincomprising more repeats provides a more potent repeat specific antibodyimmune response which may be more desirable for an effective CSP basedmalaria vaccine. A construct that contained all 38 NANP SEQ ID NO: 13repeats, CS/E, was also expressed. The expression level of CS/E washowever 5 fold lower than CS/D (FIG. 3) as demonstrated by SDS-PAGEcoomassie blue staining and densitometric analysis. Thus, CS/D offered adesired balance between bacterial yield and repeat specificimmunogenicity.

Example 3

In this example, the amino acid sequence of the native P. falciparum 3D7strain CS protein, derived from the NCBI Genbank (accession numberXM_001351086.1) was used to designed a nucleotide sequence that moreclosely match the codon usage pattern of highly expressed E. coli geneswithout changing the amino acid sequence (for example, the codon-usagetables found at http://www.biology.wustl.edu/gcg/docdata/ecohigh.cod andwww.kazusa.or.jp/codon/ may be used). The resultant gene is shown in SEQID NO: 1, FIG. 5. However, similar publically available algorithms mayalso be used. Codons (AGG, AGA, CGA, CUA, AUA, CCC, ACA, CCU, UCA, GGA,AGU, UCG, GGG) used at a frequency of <1% in E. coli were not used inthe desired sequence. One-hundred eighty (180) of the 278 rCSP codonswere changed, the high AT content of the rPfCSP gene was reduced from65% to a more balanced 50% following the design effort (FIGS. 5 and 6).The novel synthetic gene was synthesized and cloned into a modified pETbased plasmid (Darko, Angov et al. 2005) using the Xmal-Notl restrictionsites. Two HIS tags, one on the N-terminus, and one on the C-terminuswere added to the PfCSP construct to aid in purification. The CS/D genewas cloned into the pET plasmid in-frame with two vector encodedhexa-histidine tags, one at either end (HIS nucleotide SEQ ID NO: 3 andSEQ ID NO: 4 in combination with SEQ ID NO: 1; see also SEQ ID NO: 5,FIG. 5; peptide sequence SEQ ID NO: 2 in combination with two HIS tagprotein sequences SEQ ID NO: 6 and 7, see SEQ ID NO: 8).

The expression plasmid was transformed into Top 10 E. coli cells(Invitrogen, Carlsbad, Calif.) and sequenced on both strands. Theplasmid was then transformed into a series of E. coli strains includingTUNE™ (Novagen, EMD Chemicals, Gibbstown N.J.), BL21™ (New EnglandBiolabs, Ipswich, Mass.), SHUFFLE™ (New England Biolabs, Ipswich,Mass.), ROSSETTA™ (Novagen, EMD Chemicals, Gibbstown N.J.) and C43™(Lucigen, Middleton, Wis.) for expression. The SHUFFLE™ strain (NewEngland Biolabs, Ipswich Mass., described in U.S. Pat. No. 6,569,669,incorporated by reference) is engineered to form disulphide bonds in itscytoplasm (Bessette, P. H. et al. (1999) Proc. Natl. Acad. Sci. USA, 96,13703-13708). It was found that the E. coli SHUFFLE™ strain transfectedwith SEQ ID NO: 5 gave better cell growth and soluble protein expressionin non-animal based media and was therefore selected as the host strain.

Example 4

To produce laboratory grade protein product, bacteria carrying theexpression construct was grown in a number of media that do not containanimal-derived products. Several non-animal derived media (includingAPS-SELECT™ (BD, Sparks, Md.), HYPERBROTH™ (AthenaES, Baltimore Md.),and PHYTONE™ (BD, Sparks, Md.) containing Superbroth were tested forexpression of the CS/D protein in a Shuffle™ clone (a CS/D clone). Apreferred expression medium contained: Phytone™ 10 g/L, Yeast Extract 5g/L, Magnesium Sulfate heptahydrate 0.24 g/L, Ammonium Sulfate 3.3 g/L,Monobasic Potassium Phosphate 6.8 g/L, Sodium Phosphate Dibasic 7.1 g/L,Glycerol 5 ml/L, Dextrose 0.5 g/L and Kanamycin 0.1 g/L.

Typically in the laboratory, CS/D clone was grown for 16 hrs in a smallvolume of media (about 25 ml) at about 37° C. which was then inoculatedinto 1.5 L in a fermentation tank (New Brunswick Scientific). Thefermentation culture was grown for ˜6 hrs at about 37° C., pH 6.8 and400 rpm agitation to a final optical density at about 600 nm of ˜6 to 7AU. The culture was induced using 0.5 mM IPTG for 2 hrs, and the finalbacterial cell pellet was harvested by centrifugation and stored atabout −70° C.

In the laboratory, CS/D protein was purified from the bacterial cellpellet using 3 chromatographic steps, first was a Ni-affinity column(Qiagen), the second was a Q-sepharose anion exchange column (GEHealthcare) and the third was gel-filtration using a Superdex-75 column(GE Healthcare). A typical lab-scale purification was initiated byresuspending 10 g of bacterial cell paste in a resuspension buffer (20mM Phosphate buffer+450 mM NaCl+0.5% Sarkosyl (pH=7.2)) and lysing bymicrofluidization. The lysate was then cleared by centrifugation,diluted five-fold in 20 mM Phosphate buffer+450 mM NaCl (pH=7.2) andloaded on 15 ml Ni-NTA Superflow matrix (Qiagen) column mounted on aAKTA Purifier FPLC system (GE Healthcare). The column was washed with120 ml of wash buffer-1 (20 mM Phosphate buffer+450 mM NaCl+0.1%Sarkosyl+20 mM imidazole, pH=7.2) followed by 120 ml of wash buffer-2(75 mM Tris, pH 9.0). Protein was eluted from the Ni column usingNi-elution buffer (500 mM imidazole, 75 mM Tris, pH 9.0).

The elution from the Ni-column was diluted five-fold in Q-dilutionbuffer (75 mM Tris, pH 9.0) and loaded on a packed 3 ml Q-sepharosecolumn. The column was washed with 10 volumes of the Q-dilution buffer.Protein from the Q column was eluted using a linear gradient of 100%Q-dilution buffer to 100% Q-gradient buffer (75 mM Sodium Phosphatemonobasic, pH 5.0). The major peak of the protein was collected andanalyzed by SDS-PAGE.

The major protein peak of the Q column was purified further by loadingon to Superdex-75 gel-filtration matrix (GE Healthcare) packed in aVantage L Laboratory Column (Millipore). The column was pre-equilibratedin phosphate buffered saline (pH=7.2). The protein peak was monitored,collected and analyzed on SDS-PAGE.

FIG. 7 shows the results from the step-wise purification of CS/D proteinover 3 chromatographic columns Starting with 10 g bacterial paste, CS/Dprotein was purified to ˜60% purity following the Ni-NTA chromatographystep (FIG. 7A, Ni column elutions). A second step of purification on theanion exchange column resulted in ˜90% pure product (FIG. 7A, Q-columnelutions). The major protein peak from the Q column was then loaded on aSuperdex™-75 column, resulting in >95% pure protein (FIG. 7B). Theprotein was highly purified as evidenced by Silver staining (FIG. 7C).The CS/D protein band migrated as a tight band monomer undernon-reducing and reducing conditions, with a slight mobility difference,indicating the presence of reduction sensitive disulphide bonds (FIG.7C). The protein was recognized by a CSP specific mAb (FIG. 7D,western). On a western blot using polyclonal anti-sera against host cellE. coli proteins (FIG. 7D, right panel), no E. coli specific bands weredetected in the purified CS/D product (FIG. 7D, lanes 1 and 2),establishing the identity and purity of the CS/D product. The finalproduct had an endotoxin content of 0.5 EU/microgram protein as measuredby chromogenic Limulus amebocyte lysate (LAL) endpoint assay (Associatesof Cape Cod, Falmouth Mass.), which is acceptable for human-use.

Example 5

In this Example, the immunogenicity of the laboratory producedrecombinant Pf CS/D protein is shown. 2.5 micrograms of recombinant CS/Dprotein was combined with Montanide ISA 720 adjuvant (Seppic, FairfieldN.J.) or a control composition containing PBS and Montanide was used tovaccinate ten C57BL6 mice three times on days 0, 14, and 30. Thevaccines were given via intraperitoneal injection. The sera from thevaccinated mice was collected two weeks after the second and third doseand tested by ELISA to determine the level of anti-CSP antibodyresponse. FIG. 8A shows the mice exhibited a strong antibody responsefollowing the 2^(nd) and 3^(rd) doses of the vaccine. Ten control micevaccinated with PBS+Montanide showed no anti-CS antibodies.

Two weeks after the third dose, when the ELISA titer was maximal, tenCS/D vaccinated mice and ten control group mice that had received aPBS+Montanide ISA720 composition were challenged with a transgenicrodent malaria parasite P. berghei expressing a functional P. falciparumCSP gene (Pb-Pf transgenic) (Tewari, R., R. Spaccapelo, et al. (2002).The mice were challenged with about 15,000 infective Pb-Pf transgenicsporozoites via intravenous tail vein injection. Appearance of bloodstage parasites was monitored by daily giemsa staining (FischerScientific) for 15 days. Following challenge, none of the control micewere protected (all became slide-positive by day 7 post challenge),while eight of ten CS/D vaccinated mice showed sterile protection asindicated by no blood stage parasitemia (only two of the ten mice showedparasitemia by day 9; see FIG. 8B). The sera from these mice showedreactivity with CSP on the surface of methanol fixed sporozoites by animmuno-fluorescence assay (FIG. 8C). These data lend support to thevaccine candidacy of the CS/D protein as a future malaria vaccine. Thelaboratory grade CS/D vaccine therefore had an efficacy of 80% againstmalaria challenge in this rodent malaria experiment model.

Example 6

In this Example, a method using current Good Manufacturing Practice(cGMP, as found in CFR 21) for the manufacture of soluble rCSP proteinin animal-free media for vaccine use is demonstrated. cGMP are requiredfor the production of injectables for humans to produce substantiallypure protein preparations. A procedure to produce large quantities ofvaccine grade soluble protein was developed. First, a cell bank for theE. coli containing a nucleotide encoding the recombinant CS/D protein(CS/D#9-pETK-Shuffle) was established. The procedure involvedinoculating one baffled 500 ml Erlenmeyer flask containing 120 ml ofGrowth Medium (1.2 g Phytone, 0.6 g Yeast Extract, 0.029 g MgSO₄ 0.39 gammonium sulfate, 0.81 g Potassium Phosphate Monobasic, 0.84 g SodiumPhosphate Monobasic supplemented with 30 μg/ml Kanamycin sulfate and 1%Dextrose) with the CS/D#9-pETK-Shuffle bacteria stock. The culture wasgrown at 37±1° C. until an OD of 1.0 at 600 nm was obtained. Glycerolwas then added to the culture as a cryopreservative to a finalconcentration of 15% (v/v). The culture was aliquoted into cryovials at1.0 ml per vial and placed in controlled storage at about −70° C. forlong term storage. The final product (cell bank vials) met all of thecriteria set in compliance with the cGMP (CFR 21), which includedsterility of the medium at the beginning of the experiment and formationof cream colored colonies when cells were plated on a agar plate.Restriction digestion of DNA miniprep from the bacterial stock,confirmed that the plasmid was present in the frozen bacteria. The aboveprocess resulted in 100 vials of a Production Cell Bank of CS/Dexpressing bacterial cells. For the fermentation of CS/D clone at 300 Lscale in compliance with the cGMP, the procedure involved thawing aProduction Seed vial (stored at about −80±10° C.), and inoculating 0.9ml of this stock into 3 L growth medium (30 g Phytone, 15 g YeastExtract, 9.9 g Ammonium Sulfate, 20.4 g Potassium phosphate monobasic,21.3 g Sodium phosphate dibasic, 0.73 g MgSO₄, 15 ml glycerol, 30 gDextrose, 0.11 g Kanamycin, pH adjusted to 6.8). The OD of this starterculture reached 3.92 after 12 hours of incubation at 32° C. and 150 rpmagitation. This 3 L starter culture was used to inoculate the fermentercontaining 300 L of a growth medium containing 3 kg Phytone, 1.5 kgYeast Extract, 990 g Ammonium sulfate, 2.04 kg potassium phosphatemonobasic, 2.13 kg Sodium phosphate dibasic 73.5 g Magnesium sulfate,1.5 L glycerol (animal free), 30 ml antifoam, 750 g Dextrose, 11.5 gKanamycin sulfate, pH adjusted to 6.8. The culture is grown at 28° C.,400 rpm agitation, 299 L/min airflow and 2.3 psig pressure and the pHwas maintained at about 6.8 using 1N NaOH and 1N phosphoric acid (growthcurve is shown in FIG. 9). After the culture reached an OD of 7.2, itwas induced with 0.5 mM IPTG for 2 hours.

Approximately 4 kg of cell paste was harvested by continuous flowcentrifugation and stored frozen at about −80±10° C. The fermentationculture conditions were in compliance with cGMP, including the sterilityof the starting culture media and formation of cream colored colonies bythe bacteria plated on agar plate. The viability of the culture was2.2×10⁹ cfu/ml, with only gram negative rods observed during microscopicexamination.

For large scale purification of soluble protein, under GMP conditions,the following buffers were prepared.

1. 2×20 L of “Buffer A”—The Lysis and Nickel NTA Equilibration Buffer:15 mM Sodium Phosphate, 5 mM Potassium Phosphate, 450 mM SodiumChloride, 20 mM Imidazole (pH 7.4).

2. 20 L of “Buffer B”—Nickel NTA Wash 1 Buffer: 5 mM Sodium Phosphate, 5mM Potassium Phosphate, 450 mM Sodium Chloride, 20 mM Imidazole, 1.0%Sarcosyl, pH 6.0).

3. 20 L of “Buffer C”—Nickel NTA Wash 2 Buffer: 15 mM Sodium Phosphate,5 mM Potassium Phosphate, 450 mM Sodium Chloride, 20 mM Imidazole, 1.0%Triton X-100 (pH 6.0).

4. 20 L of “Buffer D”—Ni-NTA Wash 3: 75 mM Tris, 35 mM Imidazole (pH9.1).

5. 10 L of “Buffer E”—Ni-NTA Elution Buffer (75 mM Tris, 250 mMImidazole (pH 9.1).

6. 20 L of “Buffer F”—Q Equilibration and Wash Buffer: 75 mM Tris (pH9.1).

7. 10 L of “Buffer G”—Q Sepharose Wash 2: 75 mM Tris, 40 mM SodiumChloride (pH 9.1).

8. 10 L of “Buffer H”—Q Wash −3: 10.7 mM Sodium Phosphate, 2.5 mMPotassium Phosphate (pH 9.1).

9. 5 L of “Buffer I”—Q Sepharose Elution Buffer: 7.5 mM SodiumPhosphate, 2.5 mM Potassium Phosphate, 20 mM NaCl (pH 7.0).

10. 2×20 L of “Buffer M,”—Column Sanitization Solution: 0.2 N NaOH.

11. 10 Liters of “Solution 0”—Q Regeneration Buffer: 10 mM SodiumPhosphate, 260 mM Sodium Chloride (pH 6.5).

12. Nickel-Nitrile-Triacetic Acid (Ni-NTA) Superflow Column: 1018 mlpacked and equilibrated in “buffer A”.

13. Q Sepharose Column: 450 ml packed and sanitized with 5 columnvolumes (CV) of “Solution M, followed by 10 CV of water, 3 CV of “bufferO” and equilibrated in “buffer F”.

The following procedure was used for cGMP compliant proteinpurification.

Day 1: A total of 1.5 Kg of frozen wet cell paste from the −80° C.storage was placed into a 2-8° C. refrigerator overnight for thawing. Onday 1, about 12 L of chilled “Buffer A” (Lysis Buffer) was added to thethawed cell paste (8 ml of solution A was added per gram of cell paste).The cell paste was stirred using a Turrax homogenizer on wet ice for 20min, and the final temperature of the cell suspension was about 6.9° C.The bacterial suspension was lysed by running through a microfluidizer,and the final temperature of the lysate was about 7.6° C. Followinglysis, the solution was cleared by centrifugation in a Sorvall highspeed centrifuge at 14,500 RPM, for 60 minutes (temperature set to about2-8° C.). A total of 12.6 L of supernatant was decanted and loaded on toa 980 ml packed volume Ni-NTA column at the rate of 100 ml/min. This Niload sample was analyzed by SDS-PAGE as shown in FIG. 10, lane 1. Thecolumn was then washed with 11 L “Buffer A” at 190 ml/ml flow rate (FIG.10, lane 2), followed by 11 L of “Buffer B”, at 210 ml/ml (FIG. 10, lane3) and 13.5 L “Buffer C” at 175 ml/min (FIG. 10, lane 4). The column wasstored at about 4° C. overnight in “Buffer C”.

Day 2: The Ni column was washed with about 9.2 L “Buffer D” at 142ml/min (FIG. 10, lane 5) and the protein was eluted from the Ni-columnin 2 L of “Buffer E” at 140 ml/min flow-rate. The OD of the Ni-elutatepool was 1.136 (FIG. 10, lane 6). The protein elution from the Ni columnwas diluted 1:2 in “Buffer E” to a final OD of 0.5 and was storedovernight at about 4° C.

Day 3: The 4 L diluted Ni-NTA elution was loaded on a 486 ml Q Sepharosecolumn at 84 ml/min. The column was pre-equilibrated in Buffer F. The Qcolumn was washed with 5.5 L of “Buffer F” at 79 ml/min (FIG. 10, lane8), followed by 5.5 L of “Buffer G” at 82 ml/min (FIG. 10, lane 9), andagain with 2 L of “Buffer F” at 82 ml/min (FIG. 10, lane 10), followedby 2 L of “Buffer H” at 82 ml/min (FIG. 10, lane 11) and the finalproduct was eluted from the Q Sepharose column in 1500 ml “Buffer I” at82 ml/min flow-rate (FIG. 10, lane 12). The fractions containing theprotein were pooled (1.5 L) and optical density at 280 nm showed the ODwas 0.96. The protein was then diluted to an OD of about 0.4, finalvolume 3.57 L, which amounted to 1,775 micrograms of CS/D solubleprotein.

Day 4: The protein was filtered through a 0.2 micron cellulose acetatefilter and frozen down at about −70° C. as purified bulk soluble CS/Dprotein (FIG. 10, lane 13).

The purity and stability of the cGMP grade CS/D protein wascharacterized as follows. Evidence of CS/D protein homogeneity wasdetected by coomassie blue stained SDS-PAGE, with samples run undernon-reduced and reduced conditions (FIG. 11). Note that up to 8micrograms of cGMP grade protein was loaded to check for product purity.

Silver stained bulk purified CS/D protein was also analyzed underreducing condition, and it also showed a highly purified product, asdemonstrated in FIG. 12. Note that up to 2 microgram protein was loadedto check for purity (FIG. 12).

TABLE 1 Sample Test Specification Result Pass/Fail Purified Bulk LotHomogenous Clear, PASS Bulk Evaluation clear, colorless colorless liquidPurified Sterility No growth No growth PASS Bulk Purified pH 6.9-7.5 7.3PASS Bulk Purified Protein Content Report as Tested 418.9 PASS Bulk byBCA 400 μg/ml μg/ml Purified Identity by Report as Tested ~40 PASS BulkSDS-PAGE Molecular weight kDa (reduced) between 35-50 kDa PurifiedRabbit Pyrogen Non- Non PASS Bulk pyrogenic pyrogenic Purified Purity byPurity >90% >99% PASS Bulk SDS-PAGE

Table 1 demonstrates a summary of the testing and results of thepurified bulk CS/D protein expressed and purified by the above-describedcGMP method. The purified protein fulfills and exceeds the requirementsfor a human-grade vaccine injectable.

Host cell protein (HCP) content of cGMP grade CS/D was measured byELISA. The cGMP product contained <1 ng/ml of HCP in a 418 microgram perml protein concentration bulk as measured by Cygnus HCP ELISA. Thus, theproduct is about >99% pure. No endotoxin was detectable by LAL assay(minimum detection limit is 0.1 EU/ml) or by the Gel-Clot endotoxinassay. The product is suitable as a human-use injectable.

Stability of CS/D product during freeze-thaw was determined as follows:The cGMP bulk CS/D protein was thawed and run on a gel under reduced andnon-reduced conditions. One way to detect if the protein is unstable insolution is to analyze it on a gel before and after spinning out anyprecipitated material which can removed by centrifugation. FIG. 13 showsno difference in band intensity in pre and post-spin samples of the bulkprotein CS/D freshly thawed and analyzed under reduced or non-reducedconditions. This indicated that no rCSP would be lost to precipitationduring the freeze-thaw. It should be noted that western blot is a verysensitive way to detect protein aggregation and we did not see any rCSPsmear above the main band.

Thermal stability of the CS/D product was evaluated as follows. Inaddition to loss during freeze-thaw, the thermal stability of a CS/Dproduct would be an important characteristic, particularly if additionalsteps of chemical modification are to be carried out, under an elevatedtemperature. Thermal stability experiments with the CS/D product wereconducted at four different temperatures. It was found that the proteindid not degrade or aggregate between −70 to +22° C. incubation for up to96 hrs as evidenced by non-reduced SDS-PAGE (FIG. 14). At 37° C., someself aggregation was observed (still only distinct bands of multimerswere seen and not a smear). Some degradation was also evidenced byreduction in the density of the main band following >60 hr of incubationat 37° C. However, the CS/D product was thermally stable between −70° C.and +22° C.

Reversed Phase HPLC analysis of the cGMP rCSP product was used to assessconformational homogeneity. The CS/D bulk protein was analyzed on areversed-phase C18 column (Protein and Peptide™ VYDAC) to determineconformational homogeneity in solution. As seen in the elution profile(FIG. 15), the protein eluted as a single peak and all minor peaks werealso present in the PBS control run. Hence, the majority of the CS/Dproduct is present in a single conformation in the bulk protein.

Immunogenicity and challenge data with the cGMP CS/D product weredetermined as follows. The cGMP CS/D protein product was combined withMontanide ISA 720 adjuvant (Seppic, Fairfield N.J.) and vaccinatedthrice into ten C57BL6 mice, two weeks apart. Each mouse received 5micrograms of the antigen and 70 microliters of adjuvant per dosesubcutaneously. Control mice received the adjuvant alone in PBS. Thesera from the vaccinated mice was collected two weeks after the secondand third dose and tested by ELISA. Mice were then challenged with atransgenic strain of P. berghei parasites expressing a functional P.falciparum CSP gene (Tewari, Spaccapelo et al., 2002). A dose of 2500transgenic sporozoites was given intravenously, three weeks after thethird vaccine dose. All mice that received the CS/D vaccine developedantibodies to the protein as measured by a repeat specific ELISA (FIG.16). Antibodies were boosted by the third vaccination as seen by higherP3 vs. P2 titers. Seven of 10 CSP vaccinated animals did not becomeinfected following challenge, as evidenced by negative blood smears upto day 12 post challenge. All the adjuvant control mice showed bloodstage parasites by day 4 post challenge (FIG. 17B). There was a positivecorrelation between repeat specific ELISA titers and full-length proteinELISA titers. However all of the protected animals did not have hightiter antibodies (FIG. 18). Thus the cGMP grade CS/D vaccine had anefficacy of 70% in this rodent malaria experimental model.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications are specifically and entirely incorporated by reference. Asused in this specification, the singular forms “a”, “an”, and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a recombinant CSP” includes one ormore of such circumsporozoite proteins. A reference to “an adjuvant”includes one or more of such adjuvants, and so forth. It is intendedthat the specification and examples be considered exemplary only withthe true scope and spirit of the invention indicated by the followingclaims. Furthermore, where the term “comprising of” appears, it iscontemplated that the terms “consisting of” or “consisting essentiallyof” could be used in its place to describe certain embodiments of thepresent technology.

REFERENCES

-   Blum-Tirouvanziam, U., C. Beghdadi-Rais, et al. (1994). “Elicitation    of specific cytotoxic T cells by immunization with malaria soluble    synthetic polypeptides.” J Immunol 153(9): 4134-4141.-   Blum-Tirouvanziam, U., C. Servis, et al. (1995). “Localization of    HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein    of Plasmodium falciparum.” J Immunol 154(8): 3922-3931.-   Coppi, A., C. Pinzon-Ortiz, et al. (2005). “The Plasmodium    circumsporozoite protein is proteolytically processed during cell    invasion.” J Exp Med 201(1): 27-33.-   Darko, C. A., E. Angov, et al. (2005). “The clinical-grade    42-kilodalton fragment of merozoite surface protein 1 of Plasmodium    falciparum strain FVO expressed in Escherichia coli protects Aotus    nancymai against challenge with homologous erythrocytic-stage    parasites.” Infect Immun 73(1): 287-297.-   Doolan, D. L., H. P. Beck, et al. (1994). “Evidence for limited    activation of distinct CD4+ T cell subsets in response to the    Plasmodium falciparum circumsporozoite protein in Papua New Guinea.”    Parasite Immunol 16(3): 129-136.-   Doolan, D. L., R. A. Houghten, et al. (1991). “Location of human    cytotoxic T cell epitopes within a polymorphic domain of the    Plasmodium falciparum circumsporozoite protein.” Int Immunol 3(6):    511-516.-   Doolan, D. L., C. Khamboonruang, et al. (1993). “Cytotoxic T    lymphocyte (CTL) low-responsiveness to the Plasmodium falciparum    circumsporozoite protein in naturally-exposed endemic populations:    analysis of human CTL response to most known variants.” Int Immunol    5(1): 37-46.-   Doolan, D. L., A. J. Saul, et al. (1992). “Geographically restricted    heterogeneity of the Plasmodium falciparum circumsporozoite protein:    relevance for vaccine development.” Infect Immun 60(2): 675-682.-   Tewari, R., R. Spaccapelo, et al. (2002). “Function of region I and    II adhesive motifs of Plasmodium falciparum circumsporozoite protein    in sporozoite motility and infectivity.” J Biol Chem 277(49):    47613-47618.-   Tewari, R, D. Rathore et al. (2005) “Motility and infectivity of    Plasmodium berghei sporozoites expressing avian Plasmodium    gallinaceum circumsporozoite protein.” Cell Microbiol. 2005    (5):699-707.-   Vaughan, K., M. Blythe, et al. (2009). “Meta-analysis of immune    epitope data for all Plasmodia: overview and applications for    malarial immunobiology and vaccine-related issues.” Parasite Immunol    31(2): 78-97.-   Zavala, F., A. H. Cochrane, et al. (1983). “Circumsporozoite    proteins of malaria parasites contain a single immunodominant region    with two or more identical epitopes.” J Exp Med 157(6): 1947-1957.-   Zevering, Y., C. Khamboonruang, et al. (1994). “Life-spans of human    T-cell responses to determinants from the circumsporozoite proteins    of Plasmodium falciparum and Plasmodium vivax.” Proc Natl Acad Sci    USA 91(13): 6118-6122.

The invention claimed is:
 1. An anti-malaria vaccine comprising: arecombinant Plasmodium falciparum circumsporozoite protein (rCSP) thatcomprises SEQ ID NO:2, or an rCSP of 278 amino acids in length having95% sequence identity to SEQ ID NO:2; and one or more adjuvants.
 2. Thevaccine of claim 1, wherein the adjuvant is a water-in-oil emulsioncomprising a metabolizable oil.
 3. The vaccine of claim 1, wherein thevaccine has an endotoxin level less than about 5 endotoxin units permicrogram of protein.
 4. The vaccine of claim 1, wherein the vaccinecomprises less than about 1 ng/ml of bacterial host proteins.
 5. Thevaccine of claim 1, wherein the vaccine has a protein content, and theprotein content is greater than 95% recombinant P. falciparum CSP asmeasured by gel densitometry.
 6. The vaccine of claim 1, wherein theprotein content is at least about 99% recombinant P. falciparum CSP asmeasured by gel densitometry.
 7. A method of eliciting an immuneresponse against malaria in an animal or human comprising administeringthe vaccine of claim 1 to the animal or human.
 8. The method of claim 7,wherein the vaccine is administered intramuscularly.
 9. The vaccine ofclaim 1, wherein the rCSP comprises 18 or 19 NANP (SEQ ID NO: 13)repeats.
 10. The vaccine of claim 9, wherein the rCSP comprises 19 NANP(SEQ ID NO: 13) repeats.
 11. The vaccine of claim 9, wherein the rCSPfurther comprises 0 to 3 NVDP (SEQ ID NO: 14) repeats.
 12. The vaccineof claim 9, wherein the rCSP further comprises 3 NVDP (SEQ ID NO: 14)repeats.
 13. The vaccine of claim 1, wherein the vaccine comprises arecombinant P. falciparum CSP that lacks Met₁, to Cys₂₅ of theN-terminal region of native P. falciparum circumsporozoite protein. 14.The vaccine of claim 1, wherein the vaccine further comprises a rCSPhaving a C-terminal region that lacks ten to fourteen C-terminus aminoacid residues of native P. falciparum circumsporozoite protein.
 15. Thevaccine of claim 1, wherein the rCSP comprises SEQ ID NO:8.
 16. Thevaccine of claim 1, wherein the adjuvant is provided in the form of aparticle.
 17. The vaccine of claim 16, wherein particle is selected fromthe group consisting of a microparticle or a liposome that comprises atleast one adjuvant inside the microparticle or liposome, or on thesurface of the microparticle or liposome.
 18. The vaccine of claim 1,wherein the rCSP is covalently linked to a particle.
 19. The vaccine ofclaim 18, wherein the particle is selected from the group consisting ofa microparticle that comprises at least one adjuvant inside or on thesurface of the microparticle and a liposome that comprises at least oneadjuvant inside or on the surface of the liposome.
 20. The method ofclaim 7, wherein the method induces a broad antibody response againstmalaria in the animal or human.